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Biochemistry (Moscow)

, Volume 78, Issue 5, pp 455–462 | Cite as

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

  • I. M. Vikhlyantsev
  • A. D. Okuneva
  • U. V. Shumilina
  • N. N. Salmov
  • A. G. Bobylev
  • N. V. Molochkov
  • Z. A. Podlubnaya
Article
  • 120 Downloads

Abstract

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca2+-depleting solution before their homogenization to decrease activity of Ca2+-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca2+-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ∼5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ∼30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca2+-dependent proteases.

Key words

striated muscles of mammals NT- N2A- N2BA- and N2B-isoforms of titin T2-fragments actin-activated ATPase activity of myosin Ca2+-depleting solution 

Abbreviations

ATPase

adenosine triphosphatase

CD

circular dichroism

CaD-solution

Ca2+-depleting solution

LV

left ventricle

MHC

myosin heavy chains

μ

ionic strength

S1

myosin subfragment 1

SR

sarcoplasmic reticulum

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Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • I. M. Vikhlyantsev
    • 1
  • A. D. Okuneva
    • 1
    • 2
  • U. V. Shumilina
    • 1
  • N. N. Salmov
    • 1
  • A. G. Bobylev
    • 1
  • N. V. Molochkov
    • 1
  • Z. A. Podlubnaya
    • 1
    • 2
  1. 1.Institute of Theoretical and Experimental BiophysicsRussian Academy of SciencesPushchino, Moscow RegionRussia
  2. 2.Pushchino State Institute of Natural SciencesPushchino, Moscow RegionRussia

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